Therapeutic strategies are being sought to block HIV-1 replication in vivo. One of the most attractive approaches currently being tested is the use of viral protease inhibitors. These compounds are highly effective in blocking viral replication in cell culture with little evidence of toxicity. In anticipation of protease inhibitors being used in the near future in a clinical setting, I propose to explore the potential for the appearance of protease inhibitor-resistant HIV-1. Drug-resistant viruses have been found after treatment with four small molecule inhibitors, indicating the potential for the development of such a virus. The proposed research will result in the identification of a potential limitation to this new form of therapy, develop techniques for identifying the presence of protease inhibitor-resistant viruses, and examine the molecular basis of resistance. The protease inhibitor A-77003 from Abbott Laboratories will be used to select for inhibitor-resistant virus in cell culture. This compound is currently in preclinical testing. Variant viruses will be selected starting with a cloned virus and also using a series of patient isolates. In addition, the extent of sequence variability within the protease domain will be increased prior to selection by using our large collection of protease mutants. A combination of these approaches will lead to the identification of inhibitor-resistant virus. Such variants will be molecularly cloned, the relevant sequence change(s) identified, the biochemical effects of this change on the viral protease determined, and the potential for cross-resistance examined. A rapid screen to identify sequence changes associated with resistance will be developed. This work is preparatory to obtaining virus isolates from patients being treated with A-77003.